Over 30 million men suffer from erectile dysfunction in the United States. Constriction and dilation of the cavernosal vasculature determines penile erection. In the absence of arousal stimuli, Ca2+-sensitizing RhoA/Rho-kinase signaling maintains vasoconstriction, keeping the penis non-erect. Upon arousal, nitric oxide (NO), released from nerves and endothelial cells, induces dilation and erection. Although NO stimulates erection, the cellular mechanism of NO is unknown. NO binds soluble guanylate cyclase to stimulate an increase in cyclic GMP (cGMP) and the subsequent activation of cGMP-dependent protein kinase (cGK). In the penis, cGK has been proposed to induce dilation through activation of membrane K+ channels to cause hyperpolarization, inhibition of membrane Ca2+ channels to decrease activator Ca+, and stimulation of sarcoplasmic reticular Ca2+ uptake to sequester the cation. However, recent work suggests that high levels of activator Ca2+ are not maintained during constriction and it is the Ca2+-sensitizing effect of RhoAJRho-kinase that must be overcome to cause dilation. We hypothesize that cGK inhibits RhoA translocation to the membrane leading to a reduction in Rho-kinase activity and removal of its inhibitory action on myosin light chain (MLC) phosphatase. This dis-inhibition leads to reduced MLC phosphorylation, smooth muscle relaxation and erection. We further hypothesize that the long-term expression of components of the RhoA/Rho-kinase signaling pathway are inversely related to NO bioavailability. These hypotheses will be tested by 3 specific aims: 1) to determine if NO/cGMP/cGK signaling antagonizes RhoA activation to evoke dilation and penile erection; 2) to determine if gene transfer of endothelial nitric oxide synthase (eNOS) to the penis will down-regulate RhoA/Rho-kinase signaling to augment erection; and 3) to determine if reduced NO bioavailability (pharmacological blockade and denervation) leads to up-regulation of the RhoA/Rho-kinase pathway and erectile dysfunction. The approach will utilize rat and mouse models of erection. The experiments will determine the effect of NO/cGMP/cGK on biochemical, pharmacological and physiological measures of the RhoA/Rho-kinase pathway in the intact penis and in isolated cavernosal strips. Gene transfer of dominant-negative RhoA and endothelial NOS to the penis will provide a powerful tool to manipulate the activity of the RhoA/Rho-kinase pathway. Contractile force measurements in isolated cavernosal strips (intact and permeablizied) will provide evidence for Ca2+ sensitization and its regulation by cGMP/cGK. These studies will define the molecular basis for NO-mediated cavernosal vasodilation in the normal state and how long-term changes in the Ca 2+ sensitizing mechanism contribute to erectile dysfunction. [unreadable] [unreadable]